1. Field of the Invention
The present invention generally relates to multidimensional imaging. More particularly, the invention relates to three-dimensional (3D) microscopy obtained by varying focus during acquisition of an image followed by image deconvolution.
2. Background
The rate of acquisition of 3D imaging (e.g., acquisition of fluorescent signals in 3D) using digital imaging-based microscopy is a critical factor in a number of applications. Because the depth of focus is relatively shallow and becomes shallower as the resolving power of the objective lens increases (as the numerical aperture increases), it is often necessary to acquire image samples at multiple levels of focus in the object of interest in order to avoid missing potentially important sources of signal. In applications requiring large sample sizes, the rate of acquisition sets an upper limit on the possible throughput. In applications where the object of interest moves during acquisition, this rate correlates directly with the amount of motion blur in the acquired images.
Current practice is to acquire individual image “slices” at discrete sampling planes by moving focus between each acquisition, then conceptually to combine these slices into image stacks (“volumes”) which are processed further as needed. This procedure introduces several bottle-necks: (1) quickly changing focus introduces vibrations in the sample that degrade the image, and waiting for the vibrations to dampen or slowly moving focus slows the process, (2) images must be collected, digitized, and transmitted individually—each step introducing delays, and (3) processing of the 3D data sets to remove out-of-focus blur is computationally burdensome. What is needed is a set of techniques that would allow for rapid and reliable 3D acquisition of data.
The referenced shortcomings are not intended to be exhaustive, but rather are among many that tend to impair the effectiveness of previously known imaging techniques; however, those mentioned here are sufficient to demonstrate that methodology appearing in the art have not been altogether satisfactory and that a significant need exists for the techniques described and claimed in this disclosure.